Genetic Variants, Environmental Factors And The Gene- Environmental Interactions In Association Of Children Attention Deficit Hyperactivity Disorder

Objectives:1.To investigate the environmental factors of attention deficit hyperactivity disorder（ADHD）of children in order to to provide evidence for ADHD prevention.2.We performed a pathway-based analysis to test individual SNPs and the overall evidence of genetic polymorphisms involed in circadian pathway in association with ADHD susceptibility.3.We idendified the association between the genes involved in the dopaminergic,serotonergic and noradrenergic pathways and ADHD susceptibility for children.4.To explore the potencial gene-environment interaction of environmental factors and genetic liability on children ADHD,and to provide the way for further research on the pathogenesis of ADHD.Methods:ADHD children and health children were enrolled from the Liuzhou Women and Children Healthcare Hospital and two primary schools in Liuzhou according to standardized procedures of ADHD assessment and exclusion criteria.After having get the permission for the eligible cases and controls to participate in our study and informed consent,we collected ADHD related information and blood samples during December 2015 to May 2016.As a total,168 cases and 233 controls（6-12 years of age）were recruited.1.In this case-control study,epidemiological survey was carried out to investigate the influencing factors of children ADHD.Univariate and multivariate unconditional logistic regression analysis were conducted to evaluate the risk factors of ADHD for children.2.In the second and third part,numbers of candidate single nucleotide polymorphisms（SNPs）involed in circadian pathway,dopaminergic,serotonergic and noradrenergic pathways were genotyped using MassARRAY genotyping platform.Hardy-Weinberg equilibrium（HWE）for genotypes was calculated in controls by a goodness-of-fit chi-squared test.Logistic regression analyses were applied to estimate the effect of individual SNPs on ADHD susceptibility by computing odds ratios（ORs）and 95%confidence intervals（95%CIs）under assuming variant alleles as risk alleles,and four inheritance models（codominant,dominant,additive and recessive models）were estimated for each SNP.Classification and regression trees（CART）analysis was growed to explore the potential gene-gene interactions among the candidate genes and cumulative analysis was performed to assess the aggregated effect of the potentially multiple genetic variants.3.In the fourth part,the gene×environment interaction between the SNPs involved in PER1,BMAL2、ADRA2A、DRD2 and SCL6A4 genes and in-utero smoking exposure and parenting style was calculated.Multiplicative interaction was evaluated by the interaction term of logistic regression model and additive interaction was calculated using the model made by Andersson et al.Result:1.Compared with authoritative parenting style,the indulgent and neglectful parenting style and inconsistent parenting style showed a risk association with ADHD,separately（OR:7.67,95%CI:3.36-17.47,P=0.003;OR:2.87,95%CI:1.43-5.77,P<0.001）.The results of univariate unconditional logistic regression analysis showed that maternal mood stress during pregnancy and maternal age were association with ADHD.Maternal age is a protective factor for ADHD（OR=0.95,95%CI:0.91-0.99,P=0.028）,and maternal mood stress during pregnancy is associated with increased risk of ADHD（OR=19.21,95%CI:2.42-150.41,P<0.001）.However,the association between maternal age and ADHD was disappeared in the multivariate unconditional logistic regression analysis.Meanwhile,in-utero smoking exposure showed a risk association with ADHD and its symptom domains.Compared with individuals who did not have the exposure of in-utero smoking,individuals with the exposure had a 1.77-fold increase in risk of ADHD（95%CI:1.14-2.76）,and individuals with the in-utero smoking exposure had a1.76-fold increase in risk of inattention subtype（95%CI:1.16-2.77）,and had a 2.80-fold increase in risk of ADHD hyperactivity.There was no statistical difference between ADHD patients and controls in levels of blood lead level,preterm birth,and maternal diseases during pregnancy.2.The results of candidate SNPs of circadian pathway association study:in the single-loci analysis,we identified a new polymorphism–PER1 rs2518023–to be in a significant association with ADHD susceptibility(P_FDR=0.036).In addition,a variant of BMAL2 rs2306074 showed statistical significant at the uncorrected 0.05significance level,but it was not survived after the FDR adjustment.The following CART analysis was performed to test the multi-loci interactions of circadian clock genes in association with ADHD.The results revealed potential gene-gene interactions among PER1,BMAL2 and NR1D1 variants.Individuals carrying combination of PER1rs2518023 GG or GT,BMAL2 rs2306074 TC or TT NR1D1 rs939347 GA or AA genotypes displayed the highest risk associated with ADHD compared with other groups.The cumulative effect of unfavorable alleles of PER1 rs2518023 G allele,BMAL2 rs2306074 T allele was found.We calculated the total number of unfavorable alleles for each subject and set subjects with 0 unfavorable alleles as the reference group.We found that the risk of ADHD increased significantly along with the increasing number of unfavorable alleles（OR=1.40;95%CI:1.19-1.90,P=0.001）.3.The results of candidate SNPs of dopaminergic,serotonergic and noradrenergic pathways association study:We identified ADRA2A rs553668,DRD2 rs1124491 and SLC6A4 rs6354 to be in a significant association with ADHD susceptibility（P<0.05）in the single-loci analysis,but only SLC6A4 rs6354 survived after the FDR adjustment.The results revealed a potential gene-gene interactions among ADRA2A rs553668 and DRD2 rs1124491 variants.The cumulative effect of unfavorable alleles of ADRA2A rs553668 G,DRD2 rs1124491 G allele and SLC6A4 rs6354 G alleles was found.The total number of unfavorable alleles for each subject was calculated and set subjects with0¹ unfavorable alleles as the reference group.We found that the risk of ADHD increased significantly along with the increasing number of unfavorable alleles（OR=1.42,95%CI:1.15-1.77,P=0.001）.4.The results of gene-environmental interaction:the multiplicative and additive interactions were observed between the in-utero smoking exposure and the BMAL2rs2306074 and ADRA2A rs553668 in relation to ADHD separately with regard to ADHD for children.The risk of ADHD for children carrying BMAL2rs2306074 G allele or ADRA2A rs553668 G allele with in-utero smoking exposure showed significantly increase compared to those without in-utero smoking exposure.Moreover,the multiplicative gene-environment interactions were observed between DRD2rs1124491,ADRA2A rs553668 polymorphism and indulgent and neglectful parenting style,but no additive interactions were detected between them.Conclusions:1.Maternal mood stress during pregnancy,maternal smoking exposure during pregnancy,and indulgent and neglectful parenting style parenting style were identified to be the potential environmental risks to children ADHD.Therefore,health education during pregnancy should be strengthened in order to avoid the joint effects of risk environmental factors on the children ADHD.2.The results of the current study reveal the important role of genetic variation of circadian rhythm system to the susceptibility of children ADHD.3.The findings of the current pathway-based analysis indicate a potencial genetic mechanism for children ADHD,providing a new way to research of ADHD pathogenetic mechanism.4.The findings of gene-environmental interactions analyses suggested that the environmental factors such as in-utero smoking exposure might superimpose and promote the susceptibility of ADHD.